The importance of gap junction (GJ) in hearing is highlighted by the finding that mutations in at least 4 subtypes of connexins (Cxs, which are building blocks of GJs) result in deafness. Genes coding for Cx26 and Cx30 are the most common cause of hereditary non-syndromic deafness. It is estimated that 25-50% of prelingual childhood deafness cases are associated with mutations in Cx26, which affect millions of patients worldwide (Ahmad et al., 2003; Gerido and White, 2004). Despite the importance of Cxs in hearing, we know very little about the role Cxs play in the cochlea. Our published data show that Cx26 and Cx30 co-localize in most cochlear GJ plaques and they co-assemble to form GJs (Ahmad et al., 2003). Targeted deletion of either Cx26 or Cx30 in the mouse results in deafness (Cohen-Salmon et al., 2002; Teubner et al., 2003) demonstrating that a reduction of Cx diversity in forming cochlear GJs is sufficient to cause deafness. Our preliminary data demonstrated that deafness in Cx30 knockout (Cx30KO) mice was caused not simply by the loss of Cx30 but due to instability and under-expression of Cx26 in the cochlea. Therefore, investigations into the molecular determinants affecting the stability and degradation of cochlear Cx become therapeutically important. In this proposal we plan to test the hypothesis that cochlear heteromeric GJs consisting of Cx26 and Cx30 are more stable than homomeric GJs. We will test this hypothesis by: 1) comparing the half-life of hetero and homomeric GJs; 2) delineating the primary pathways utilized in degrading homo and heteromeric GJs. Estimation of the half-life based on the pulse-chase assay is a reliable method for the measurement of stability of proteins and will be employed to measure the half-life of cochlear GJs of different molecular configurations in vitro and in vivo. Pathways utilized to degrade GJs will be investigated to analyze whether homomeric GJs consisting of Cx26 or Cx30 degrade faster than heteromeric GJ by preferentially utilizing a faster degradation pathway (the proteasomal pathway). Cxs are exceptionally-well regulated and have very short half-life (2-4 h). The results from this study will provide better understanding of molecular mechanisms and cellular factors influencing the half-life of cochlear Cxs. In Cx26 or Cx30 mutation patients, one of the two Cx genes is expected to be still intact in the genome. Investigations into the mechanisms for stabilizing the expression and slowing down the degradation of the remaining Cx gene may open up entirely new research direction for studying the deafness. [unreadable] [unreadable] [unreadable]